This application directs itself at further elucidating the molecular mechanisms regulating prestalk and prespore differentiation in Dictyostelium. The two cell types are spatially localized in the anterior (prestalk) and posterior (prespore) of the migrating slug. Signals of cAMP transduced through cell surface receptors activate the expression of genes unique or predominantly expressed in either one of the two cell types. Using diverse molecular and genetic approaches, we propose to study the cAMP receptor-activated signal transduction pathway. We will purify and clone the transacting factor that controls one class of induced prestalk genes and examine its putative posttranslational modification in response to cAMP receptor activation. The cis-acting sequences and trans-acting factor(s) controlling the expression of the prestalk, cell-type-specific Dd-ras genes and a coordinately regulated, cAMP-inducible class of prespore genes will be further defined. The trans-acting factor associated with a conserved regulatory element in the prespore promoters will be purified and analyzed. Using molecular complementation with genomic libraries, we will rescue existing mutants and mutations we will generate, blocked in the expression of cAMP-induced genes. With the plasmids that provided rescue, we will clone and characterize the wild-type genes. We will investigate the molecular mechanisms regulating spatial localization of the prestalk and prespore gene expression using prestalk and prespore specific promoter/beta-gal fusions. These will be used to examine the molecular basis of an anterior to posterior gradient of prespore gene expression within the prespore zone of the slug and the role of the extracellular morphogens DIF and cAMP in establishing and maintaining this gradient. We will also examine the role of the cell cycle in regulating cell-type differentiation and the ontogeny of prestalk and prespore cells. These diverse approaches will be integrated with the goal of understanding the integrated pathways that regulate cellular differentiation in this organism.